PROJECT SUMMARY/ABSTRACT The large epidemic outbreak of Zika virus (ZIKV) in South America has taken the world by surprise. Prior to its large outbreak, ZIKV was considered a low prevalent virus, transmitted by the Aedes mosquito, and causing a few cases of mild febrile illness in humans. However, since its arrival in the American continent, probably related to human travel, the cases of ZIKV infections have exploded and the virus continues to spread through tropical and subtropical countries in the Americas. Importantly, ZIKV infections have recently been associated with neurological disorders, including microcephaly in babies born from infected pregnant mothers, and increases in Guillain-Barr syndrome in infected individuals. Practically nothing is known about the molecular pathogenesis and virulence factors of ZIKV. Our previous studies with other flaviviruses, including West Nile virus (WVN), dengue virus (DENV) and yellow fever virus (YFV) have revealed their remarkable abilities to inhibit innate immune responses by targeting type I interferon (IFN-I) signaling, although by different mechanisms. This makes these viruses more resistant to IFN-I and contributes to their virulence and host tropism. Our lab has recently found that ZIKV also efficiently inhibits IFN-I signaling in infected cells and identified that, similar to DENV, the viral NS5 protein binds to STAT2, a critical transcription factor involved in IFN-I signaling, and targets it to degradation. However, our data also indicate that STAT2-mediated degradation by ZIKV NS5 takes place by a mechanism different from that of DENV NS5. In the context of this R21 we propose to map the domains and amino acid residues in both ZIKV NS5 and STAT2 responsible for their interactions (Aim 1) and to study the impact of NS5-mediated STAT2 degradation in ZIKV replication, host response induction and in its sensitivity to the antiviral action of IFN-I in human cells (Aim 2). The mutant ZIKVs with impaired IFN-I antagonistic properties that will be generated in this R21 proposal will represent the basis for additional studies to investigate their attenuation properties in vivo and their potential use as live attenuated vaccines against ZIKV. In addition, data generated in this R21 will be critical for future studies to better understand the mechanism by how ZIKV degrades STAT2, which might result in the identification of novel targets for antiviral development.